Polymyxins are a family of antibacterial agents comprising a cyclic peptide structure composed of diaminobutryic acid moieties and various essential amino acids together with a hydrophobic tail. Polymyxins are produced in gram-positive bacteria and act primarily on gram-negative bacteria by disruption of the outer membrane of the gram-negative bacterium. Of the recognized polymyxins, Polymyxin B and Polymyxin E (colistin) are each used therapeutically, each having been shown to be active against inter alia Pseudomonas aeruginosa, Enterobacteriaceae (such as E. coli, Klebsiella pneumoniae) and Acinetobacter spp. The toxicity of polymyxins has limited their therapeutic use to topical administration and as a last line of defense against life-threatening infections caused by bacteria resistant to standard antibiotics. Colistin and Polymyxin B have been used as veterinary medicines for several decades as treatment for gram-negative gastrointestinal infections in densely populated livestock. As a result, there are increasing reports of bacterial resistance.
The rise in multi-drug resistant bacteria, thought to be primarily as a result of antibiotic over-prescribing in humans and especially over-use in domestic animals (including prophylactic prescribing), highlights the need to moderate the use of last resort treatments such as the polymyxins. A recent report by the European Medicines Agency (2013) recommends rational antimicrobial therapy including cessation of both use in control and treatment of Salmonella spp. in poultry and pigs, and in production enhancement e.g. increase of feed efficiency or growth promotion. Furthermore, the European Agency for the Evaluation of Medicinal Products (2002) and the Codex Alimentarius Commission have set the maximum residue limit (MRL) for Colistin in various animal tissues at 50-300 μg/kg. The implementation of such requirements and recommendations requires Colistin and Polymyxin B testing methods.
An ELISA for the quantification of Polymyxin B sulphate in human serum is known. The immunogen used to derive the antibodies used in the immunoassay was Polymixyin B bonded to the primary amino groups of the diaminobutyric moieties of Polymyxin B via the crosslinking group N-(gamma-Maleimidobutyryloxy)succinimide (GMBS). The resultant antibodies were cross-reactive to Polymyxin B but not Colistin. Colistin specific antibodies are also known. There is also a commercially available ELISA which detects both Colistin and Polymyxin B using an indirect competitive immunoassay format with Colistin and Polymyxin B spotted on a microtitre plate. The antigens Colistin and Polymyxin B are both spotted on the microtitre plate, which implies that the assay incorporates a mixture of antibodies, one of which binds to Colistin and one of which binds to Polymyxin B.
Thus, previous immunogens used to raise antibodies to polymyxins make use of the parent Polymyxin B structure and appear to use a scattergun approach when synthesizing the hapten in which all of the five primary amine groups are open to derivatisation. This non-targeted approach, in which all of the primary amino groups are liable to bonding, may be the reason that the subsequently raised antibody does not exhibit appreciable binding to both Polymyxin B and Colistin as it is possible that the epitopes of the target peptide are masked by the conjugated carrier proteins.
Polymyxin B and Colistin commercial formulations each exist as mixtures of chemically related compounds, with the main polymyxin variants being Polymyxin B1 and Polymyxin B2 and the main colistin variants being Colistin A and Colistin B; and, in each of which the fatty acid terminal group is varied—being either a 5-methylheptyl or 5-methylhexyl chain (see FIG. 5).